Methods for treating diabetes mellitus

ABSTRACT

In accordance with the present invention, it has been discovered that CREB binding protein (CBP) cooperates with upstream activators involved in the activation of transcription of such signal dependent transcription factors as c-Jun (responsive to phorbol ester), serum response factor, and the like. It has also been discovered that CBP can be employed in an assay to identify compounds which disrupt the ability of such signal dependent transcription factors to activate transcription. In another aspect, it has been discovered that CBP can be employed in an assay to identify new signal dependent transcription factors. In yet another aspect of the present invention, it has been discovered that CBP can be employed in an assay to identify novel co-factor protein(s) which mediate the interaction between signal dependent transcription factors and inducer molecules involved in the activation of transcription. Accordingly, the present invention provides methods for the identification of compounds which inhibit activation of cAMP and mitogen responsive genes and methods for the identification of novel signal dependent transcription factors and co-factor proteins. In still another aspect, methods employing compounds which inhibit intracellular signal-induced response pathways have been developed for the treatment of diabetes mellitus.

ACKNOWLEDGEMENT

[0001] This invention was made in part with Government support under Grant No. GM 37828 provided by the National Institutes of Health. The Government may have certain rights in this invention.

FIELD OF THE INVENTION

[0002] The present invention relates to analytical methods. In a particular aspect, the present invention relates to methods for the identification of compounds which mediate the interaction between signal dependent transcription factors and co-factor protein(s) involved in the activation of transcription. In another aspect, the present invention relates to methods for the identification of new signal dependent transcription factors and inducer molecules involved in the activation of transcription. In yet another aspect, the present invention relates to methods for treating diabetes mellitus.

BACKGROUND OF THE INVENTION

[0003] Many eukaryotic genes are regulated in an inducible, cell type-specific fashion. Genes expressed in response to heat shock, steroid/thyroid hormones, phorbol esters, cyclic adenosine monophosphate (cAMP), growth factors and heavy metal ions are examples of this class. The activity of cells is controlled by external signals that stimulate or inhibit intracellular events. The process by which an external signal is transmitted into and within a cell to elicit an intracellular response is referred to as signal transduction. Signal transduction is generally initiated by the interaction of extracellular factors (or inducer molecules, i.e., growth factors, hormones, adhesion molecules, neurotransmitters, and other mitogens) with receptors at the cell surface. Extracellular signals are transduced to the inner face of the cell membrane, where the cytoplasmic domains of receptor molecules contact intracellular targets. The initial receptor-target interactions stimulate a cascade of additional molecular interactions involving multiple intracellular pathways that disseminate the signal throughout the cell.

[0004] Many of the proteins involved in signal transduction contain multiple domains. Some of these domains have enzymatic activity and some of these domains are capable of binding to other cellular proteins, DNA regulatory elements, calcium, nucleotides, lipid mediators, and the like.

[0005] Protein-protein interactions are involved in all stages of the intracellular signal transduction process—at the cell membrane, where the signal is initiated in the cytoplasm by receptor recruitment of other cellular proteins, in the cytoplasm where the signals are disseminated to different cellular locations, and in the nucleus where proteins involved in transcriptional control congregate to turn on or turn off gene expression.

[0006] Mitogenic signaling affects the transcriptional activation of specific sets of genes and the inactivation of others. The nuclear effectors of gene activation are transcription factors that bind to DNA as homomeric or heteromeric dimers. Phosphorylation also modulates the function of transcription factors, as well. Oncogenes, first identified as the acute transforming genes transduced by retroviruses, are a group of dominantly acting genes. Such genes, which are involved in cell division, encode growth factors and their receptors, as well as second messengers and mitogenic nuclear proteins activated by growth factors.

[0007] The binding of growth factors to their respective receptors activates a cascade of intracellular pathways that regulate phospholipid metabolism, arachidonate metabolism, protein phosphorylation, calcium mobilization and transport, and transcriptional regulation. Specific phosphorylation events mediated by protein kinases and phosphatases modulate the activity of a variety of transcription factors within the cell. These signaling events can induce changes in cell shape, mobility, and adhesiveness, or stimulate DNA synthesis. Aberrations in these signal-induced events are associated with a variety of hyperproliferative diseases ranging from cancer to psoriasis.

[0008] The ability to repress intracellular signal-induced response pathways is an important mechanism in negative control of gene expression. Selective disruption of such pathways would allow the development of therapeutic agents capable of treating a variety of disease states related to improper activation and/or expression of specific transcription factors. For example, in patients with non-insulin dependent diabetes mellitus (NIDDM), hyperglycemia develops, in part as a result of β cell failure secondary to chronic insulin resistance. This hyperglycemia appears to be exacerbated by hyperglucogonemia and increased hepatic gluconeogenesis. cAMP appears to be the major starvation state signal which triggers glucagon gene expression as well as transcription of PEPCK, the rate limiting enzyme in gluconeogenesis.

[0009] There remains, thus, a need in the art for selective disruption of intracellular signal-induced response pathways.

SUMMARY OF THE INVENTION

[0010] In accordance with the present invention, it has been discovered that CREB binding protein (CBP) cooperates with upstream activators involved in the activation of transcription by signal dependent transcription factors, such as c-Jun (responsive to phorbol ester), serum response factor, and the like. Accordingly, assays employing CBP have been developed for the identification of compounds which disrupt the ability of signal dependent transcription factors to activate transcription. In another aspect, assays employing CBP have been developed for the identification of new signal dependent transcription factors. In yet another aspect of the present invention, assays employing CBP have been developed for the identification of novel co-factor protein(s) which mediate the interaction between signal dependent transcription factors and inducer molecules involved in the activation of transcription. In still another aspect, an assay is provided to identify compounds which have the binding and/or activation properties characteristic of CREB binding protein. In still another aspect, methods employing compounds which inhibit intracellular signal-induced response pathways have been developed for the treatment of diabetes mellitus.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1 is a bar graph summarizing the injections described in Example 2. Each bar represents the percentage of positive cells expressing β-galactosidase from 2-3 experiments where 100-200 cells were injected in each experiment. [anti-CBP] denotes concentration of affinity purified CBP antiserum injected into cells. Right (hatched bars) indicate the percent lacZ positive cells after microinjection of CRE-lacZ reporter with CBP antiserum (anti-CBP) or control IgG (RbIgG). Preincubation of antisera with CBP peptide or non-specific ILS peptide (1 mg/ml) was carried out as indicated.

[0012]FIG. 2 is a bar graph summarizing the results of CBP antisera injections, as described in Example 3. Bars represent the percentage of lacZ positive (blue) cells (mean±standard deviation) from 3-5 experiments where 100-200 cells were injected in each experiment. Injected cells were identified by immunofluorescence and/or lacZ staining. Reporter plasmid encoding the lacZ reporter was microinjected into NIH3T3 cells. CRE-, SRE-, TRE-lacZ reporter activities were determined after microinjected cells were treated as described herein. CMV-, RSV-, and SV40-lacZ reporter activities were measured in the absence of inducers. Hatched bars indicate % blue cells after microinjection with CBP antiserum. Solid bars indicate % blue cells following injection with control rabbit IgG (RbIgG).

DETAILED DESCRIPTION OF THE INVENTION

[0013] Cyclic AMP (cAMP) regulates the transcription of numerous genes through protein kinase-A (PK-A) mediated phosphorylation, at Ser133, of transcription factor CREB. Within the CREB protein, a 60 amino acid Kinase Inducible Domain (KID) mediates transcriptional induction by PK-A. Based on recent work describing a nuclear CREB Binding Protein (CBP), it has been examined whether CBP is necessary for cAMP regulated transcription. Within CBP, a CREB binding domain has been identified, referred to as KIX which specifically interacts with the phosphorylated KID domain of CREB. About 600 A of solvent accessible surface area in each protein is directly involved in formation of CREB:CBP complex. Phosphorylated Ser133 coordinates with a single arginine residue (Arg-600). The apparent Kd of the CREB:CBP complex is 0.4 μM.

[0014] Antisera against CBP have been found to completely inhibit transcription from a cAMP responsive promoter, but not from constitutively active promoters. Surprisingly, CBP has also been found to cooperate with upstream activators involved in phorbol ester and serum responsive transcription. It is demonstrated herein that recruitment of CBP to certain inducible promoters is intimately involved in transmitting inductive signals from phosphorylated, and thus activated, upstream factors to the RNA polymerase II complex. A number of analytical uses for CBP and CBP-like compounds based on these observations are described herein.

[0015] In accordance with the present invention, there is provided a method for the identification of a compound which inhibits activation of cAMP and mitogen responsive genes, said method comprising:

[0016] monitoring expression of reporter in response to exposure to said compound, relative to expression of reporter in the absence of said compound,

[0017] wherein exposure to said compound is carried out in the presence of:

[0018] a signal dependent transcription factor,

[0019] a polypeptide comprising at least amino acid residues 461-661 of the protein set forth in SEQ ID NO:2, and

[0020] a reporter construct comprising a reporter gene under the control of said signal dependent transcription factor.

[0021] As employed herein, the phrase “cAMP and mitogen responsive genes” refers to early response genes which are activated in response to a diverse array of agents including mitogens, such as, growth factors, differentiation inducers and biomodulators. Examples of such agents include insulin-like growth factor (IGF-1), erythropoietin (EPO), nerve growth factor (NGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) transforming growth factor β (TGFβ), interferon, tumor necrosis factor (TNF), interleukins, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, prolactin, serotonin, angiotensin, bombesin, bradykinin, noradrenalin, putrescine, concanavalin A, various oncogenic agents including tumor viruses, UV irradiation, estrogen, progesterone, testosterone, glucagon, PEPCK and the like.

[0022] Signal dependent transcription factors contemplated for use in the practice of the present invention include phosphorylation dependent activators such as CREB, Jun, Fos, and other early response genes such as Myc, Myb, erbA, and Rel, serum responsive factor, Elk, as well as steroid hormone receptors (e.g., glucocorticoid receptor (GR)), and the like.

[0023] Polypeptides employed in the invention assay function as co-factors by binding to the signal dependent transcription factor as a necessary component of a transcriptionally active complex. Examples of such co-factors include CBP (i.e., substantially the entire amino acid sequence set forth in SEQ ID NO:2), a polypeptide comprising amino acid residues 1-661 as set forth in SEQ ID NO:2, as well as functional fragments thereof, e.g., residues 461-661, and homologues thereof, such as those identified by the method described herein for the identification of compounds which have the binding and/or activation properties characteristic of CREB binding protein. In accordance with one embodiment of the present invention, there are provided purified and isolated polypeptides, CBPs, that bind to a specific sequence within phosphorylated CREB.

[0024] As used herein, the term “purified” means that the molecule is substantially free of contaminants normally associated with a native or natural environment. CREB binding protein, or functional fragments thereof, useful in the practice of the present invention, can be obtained by a number of methods, e.g., precipitation, gel filtration, ion-exchange, reversed-phase, DNA affinity chromatography, and the like. Other well-known methods are described in Deutscher et al., Guide to Protein Purification: Methods in Enzymology Vol. 182, (Academic Press, 1990), which is incorporated herein by reference.

[0025] Alternatively, a purified CBP, or functional fragment thereof, useful in the practice of the present invention, can also be obtained by well-known recombinant methods as described, for example, in Ausubel et al., Current Protocols in Molecular Biology (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. 1993), also incorporated herein by reference. An example of recombinant means to prepare CBP, or functional fragments thereof, is to express nucleic acid encoding CBP, or functional fragment thereof, in a suitable host cell, such as a bacterial, yeast or mammalian cell, using methods well known in the art, and recovering the expressed protein, again using methods well known in the art.

[0026] CBPs, and biologically active fragments thereof, useful in the practice of the present invention can also be produced by chemical synthesis. Synthetic polypeptides can be produced using Applied Biosystems, Inc. Model 430A or 431A automatic polypeptide synthesizer and chemistry provided by the manufacturer. CBP, and biologically active fragments thereof, can also be isolated directly from cells which have been transformed with the expression vectors described below in more detail.

[0027] The present invention also encompasses nucleic acids encoding CBP and functional fragments thereof. See, for example, SEQ ID NO:1. This invention also encompasses nucleic acids which encode substantially the entire amino acid sequence set forth in SEQ ID NO:2 (for example, the nucleic acid sequence set forth in SEQ ID NO:1, as well as nucleic acid sequences which differ from that set forth in SEQ ID NO:1 due to the degeneracy of the genetic code), nucleic acids which encode amino acid residues 1-661, as set forth in SEQ ID NO:2, nucleic acids which encode amino acid residues 461-661, as set forth in SEQ ID NO:2, as well as nucleic acids which encode substantially the same amino acid sequences as any of those referred to above, but which differ only by the presence of conservative amino acid changes that do not alter the binding and/or activation properties of the CBP or CBP-like polypeptide encoded thereby.

[0028] The invention further provides the above-described nucleic acids operatively linked to a promoter, as well as other regulatory sequences. As used herein, the term “operatively linked” means positioned in such a manner that the promoter will direct the transcription of RNA from the nucleic acid. Examples of such promoters are SP6, T4 and T7.

[0029] Vectors which contain both a promoter and a cloning site into which a piece of DNA can be inserted so as to be operatively linked to the promoter are well known in the art. Preferably, these vectors are capable of transcribing RNA in vitro or in vivo. Examples of such vectors are the pGEM series (Promega Biotech, Madison, Wis.). This invention also provides a vector comprising a nucleic acid molecule such as DNA, cDNA or RNA encoding a CBP polypeptide. Examples of additional vectors useful herein are viruses, such as bacteriophages, baculoviruses and retroviruses, cosmids, plasmids, and the like. Nucleic acids are inserted into vector genomes by methods well known in the art. For example, insert and vector DNA can both be exposed to a restriction enzyme to create complementary ends on both molecules that base pair with each other and which are then joined together with a ligase. Alternatively, synthetic nucleic acid linkers that correspond to a restriction site in the vector DNA can be ligated to the insert DNA which is then digested with a restriction enzyme that recognizes a particular nucleotide sequence. Additionally, an oligonucleotide containing a termination codon and an appropriate restriction site can be ligated for insertion into a vector containing, for example, some or all of the following: a selectable marker gene, such as neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Other means are available and can readily be accessed by those of skill in the art.

[0030] Also provided are expression vectors comprising DNA encoding a mammalian CBP, or functional fragment thereof, adapted for expression in a bacterial cell, a yeast cell, a mammalian cell or other animal cell. Such vectors comprise the regulatory elements necessary for expression of the DNA in the bacterial, yeast, mammalian or animal cells. Regulatory elements are positioned relative to the DNA encoding the CBP polypeptide so as to permit expression thereof. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector includes a promoter such as the lac promoter and the Shine-Dalgarno sequence and the start codon AUG (Ausubel et al., supra 1993) for transcription initiation. Similarly a eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors can readily be obtained commercially or assembled by methods well known in the art, for example, the methods described above for constructing vectors in general. Expression vectors are useful to produce cells that express CBP or functional fragments thereof.

[0031] As employed herein, the term “reporter construct” refers to a recombinant construct, for example, an expression vector comprising a reporter gene under the control of a signal dependent transcription factor. In yet another example, the term refers to an expression vector comprising a reporter gene under the control of GAL4 response element. A compound which induces activation or inactivation of a target gene induces the reporter gene to express an exogenous identifiable “signal”. Expression of the reporter gene indicates that the target gene has been modulated. Exemplary reporter genes encode luciferase, β-galactosidase, chloramphenicol transferase, and the like.

[0032] In practicing the assays of the present invention, reporter plasmid is introduced into suitable host cells, along with CBP or a CBP-like polypeptide (or a DNA construct encoding same) and signal dependent transcription factor. The transfected host cells are then cultured in the presence and absence (as a control) of test compound suspected of being capable of inhibiting activation of cAMP and mitogen responsive genes. Next the transfected and cultured host cells are monitored for induction (i.e., the presence) of the product of the reporter gene.

[0033] In accordance with the present invention, expression of the reporter gene can be monitored in a variety of ways. Immunological procedures useful for in vitro detection of a polypeptide in a sample include immunoassays that employ a detectable antibody. Such immunoassays include, for example, ELISA, Pandex microfluorimetric assay, agglutination assays, flow cytometry, serum diagnostic assays and immunohistochemical staining procedures which are well known in the art. An antibody can be made detectable by various means well known in the art. For example, a detectable marker can be directly or indirectly attached to the antibody. Useful markers include, for example, radionuclides, enzymes, fluorogens, chromogens and chemiluminescent labels.

[0034] In accordance with still another embodiment of the present invention, there are provided methods to identify compounds which inhibit activation of cAMP and mitogen responsive genes, preferably compounds which disrupt complex comprising CREB and CBP, said method comprising:

[0035] (a) contacting a modified host cell with a test compound, wherein said modified host cell comprises:

[0036] a first fusion protein comprising a GAL4 DNA binding domain, operatively associated with the KID domain of CREB,

[0037] a second fusion protein comprising an activation domain, operatively associated with the KIX domain of CBP, and

[0038] a reporter construct comprising a GAL4 response element operatively linked to a reporter gene; and

[0039] (b) selecting those test compounds which cause reduced expression of the reporter gene product.

[0040] In a preferred embodiment of the present invention, the first fusion protein comprises a GAL4 DNA binding domain, operatively associated with CREB and/or the second fusion protein comprises an activation domain operatively associated with CBP.

[0041] As used herein, the term “disrupt” embraces compounds which cause substantially complete dissociation of the various components of the complex, as well as compounds which merely alter the conformation of one or more components of the complex so as to reduce the repression otherwise caused thereby.

[0042] Any cell line can be used as a suitable “host” for the functional bioassay contemplated for use in the practice of the present invention. Thus, cells contemplated for use in the practice of the present invention include transformed cells, non-transformed cells, neoplastic cells, primary cultures of different cell types, and the like. Exemplary cells which can be employed in the practice of the present invention include Schneider cells, CV-1 cells, HuTu80 cells, F9 cells, NTERA2 cells, NB4 cells, HL-60 cells, 293 cells, Hela cells, yeast cells, NIH3T3 cells and the like. Preferred host cells for use in the functional bioassay system are COS cells and CV-1 cells. COS-1 (referred to as COS) cells are monkey kidney cells that express SV40 T antigen (Tag); while CV-1 cells do not express SV40 Tag. The presence of Tag in the COS-1 derivative lines allows the introduced expression plasmid to replicate and provides a relative increase in the amount of receptor produced during the assay period. CV-1 cells are presently preferred because they are particularly convenient for gene transfer studies and provide a sensitive and well-described host cell system.

[0043] The above-described cells (or fractions thereof) are maintained under physiological conditions when contacted with physiologically active compound. “Physiological conditions” are readily understood by those of skill in the art to comprise an isotonic, aqueous nutrient medium at a temperature of about 37° C.

[0044] Various constructs employed in the practice of the present invention are well known in the art. Thus, the GAL4 DNA binding domain, the activation domain and GAL4 response elements have all been well characterized and extensively discussed in the art. For example, the DNA binding domain of the yeast GAL4 protein comprises at least the first 74 amino acids thereof (see, for example, Keegan et al., Science 231:699-704 (1986)). Preferably, the first 90 or more amino acids of the GAL4 protein will be used, with the first 147 amino acid residues of yeast GAL4 being presently most preferred.

[0045] Activation domains contemplated for use in the practice of the present invention are well known in the art and can readily be identified by the artisan. Examples include the GAL4 activation domain, BP64, VP16, and the like.

[0046] Exemplary GAL4 response elements are those containing the palindromic 17-mer: 5′-CGGAGGACTGTCCTCCG-3′, (SEQ ID NO:4)

[0047] such as, for example, 17MX, as described by Webster et al., in Cell 52:169-178 (1988), as well as derivatives thereof. Additional examples of suitable response elements include those described by Hollenberg and Evans in Cell 55:899 -906 (1988); or Webster et al. in Cell 54:199-207 (1988).

[0048] As used herein, the phrase “operatively associated with” means that the respective DNA sequences (represented, for example, by the terms “GAL4 response element” and “reporter gene”) are operational, i.e., work for their intended purposes; the word “functionally” means that after the two segments are linked, upon appropriate activation by a ligand-receptor complex, the reporter gene will be expressed as the result of the fact that the corresponding “response element” was “turned on” or otherwise activated.

[0049] As readily recognized by those of skill in the art, the above-described assay can be modified to facilitate identification of compounds which inhibit any of the specific interactions involved in the formation of the CREB:CBP complex.

[0050] Compounds which are capable of inhibiting activation of cAMP and mitogen responsive genes, and hence can be identified by the invention assay method, include antibodies raised against the binding domain of the protein set forth in SEQ ID NO:2, antibodies raised against the binding domain of CBP-like compounds, and the like. Presently preferred antibodies are those raised against a polypeptide fragment comprising amino acid residues from about 461 up to 661 of the protein set forth in SEQ ID NO:2; with antibodies raised against a polypeptide fragment comprising amino acid residues from about 634 up to 648 of the protein set forth in SEQ ID NO:2 (this subfragment is also set forth specifically as SEQ ID NO:3), being especially preferred. Alternatively, antibodies which are raised against the amino acid residues surrounding residue 600 of CBP (see SEQ ID NO:2) or antibodies which inhibit the phosphorylation of residue 133 of CREB are also desired (see, for example, Parker et al., Mol Cell Biol (1996) 16(2):694-703).

[0051] Antibodies contemplated for use in the practice of the present invention have specific reactivity with the above-described CBP or CBP-like compounds. Active antibody fragments are encompassed within the definition of “antibody.” As used herein “specific reactivity” refers to the ability of an antibody to recognize and bind to an epitope on CBP or CBP-like compounds. Antibodies employed in the practice of the present invention can be produced by any method known in the art. For example, polyclonal and monoclonal antibodies can be produced by methods well known in the art, as described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory 1988), which is incorporated herein by reference. The above-described CBP or CBP-like compounds can be used as the immunogen in generating such antibodies. Altered antibodies, such as chimeric, humanized, CDR-grafted or bifunctional antibodies can also be produced by methods well known to those skilled in the art. Such antibodies can also be produced by hybridoma, chemical or recombinant methodology described, for example in Ausubel et al., supra. The antibodies can be used for determining the presence of a CBP-derived polypeptide, for the purification of CBP-derived polypeptides, for in vitro diagnostic methods, and the like.

[0052] Alternative compounds which are capable of inhibiting activation of cAMP and mitogen responsive genes include polypeptide fragments comprising amino acid residues from about 461 up to 661 of the protein set forth in SEQ ID NO:2. Polypeptide fragments comprising amino acid residues set forth specifically as SEQ ID NO:3 or KIX polypeptide fragments having a mutation at residue 600 (Arg-600), set forth in SEQ ID NO:2, are preferred, while KIX polypeptide fragments substituting Gln for Arg-600 are presently most preferred. Other polypeptide fragments contemplated for use in the practice of the present invention include those comprising the KID domain, preferably those comprising residue 133 of CREB. In the most preferred CREB polypeptide fragment, serine residue 133 is mutated to an amino acid residue which can not be phosphorylated. Other compounds which inhibit CREB activity (i.e., phosphorylated-Ser133) by binding to CBP include adenovirus E1A oncoprotein (Nakajima et al. Genes Dev (1997) 11(6):738-747), and the like. Those of skill in the art will readily recognize other polypeptide fragments which will readily inhibit the formation of CREB:CBP complex employing such assays as those disclosed herein.

[0053] In accordance with another embodiment of the present invention, there is provided a method for the identification of a compound which inhibits activation of cAMP and mitogen responsive genes, said method comprising:

[0054] (1) contacting a test system with said compound under physiological conditions; and

[0055] (2) monitoring expression of reporter in response to said compound, relative to expression of reporter in the absence of said compound, wherein said reporter is encoded by a reporter construct comprising a reporter gene under the control of a signal dependent transcription factor, and

[0056] wherein said test system comprises:

[0057] said signal dependent transcription factor,

[0058] a polypeptide comprising at least amino acid residues 461-661 of the protein set forth in SEQ ID NO:2, and

[0059] said reporter construct.

[0060] In accordance with yet another embodiment of the present invention, there is provided a method for the identification of a compound which promotes activation of cAMP and mitogen responsive genes, said method comprising:

[0061] monitoring expression of reporter in response to exposure to said compound, relative to expression of reporter in the absence of said compound,

[0062] wherein exposure to said compound is carried out in the presence of:

[0063] a signal dependent transcription factor, or

[0064] a polypeptide comprising at least amino acid residues 461-661 of the protein set forth in SEQ ID NO:2, and

[0065] a reporter construct;

[0066] wherein said reporter construct comprises a reporter gene under the control of a signal dependent transcription factor.

[0067] In accordance with still another embodiment of the present invention, there is provided a method for the identification of a compound which has the binding and/or activation properties characteristic of CREB binding protein, said method comprising:

[0068] monitoring expression of reporter in response to exposure to said compound, relative to expression of reporter in the absence of said compound,

[0069] wherein exposure to said compound is carried out in the presence of:

[0070] a signal dependent transcription factor, and

[0071] a reporter construct,

[0072] wherein said reporter construct comprises a reporter gene under the control of a signal dependent transcription factor.

[0073] In accordance with a still further embodiment of the present invention, there is provided methods for the identification of a compound which has the transcription activation properties characteristic of a signal dependent transcription factor, said method comprising:

[0074] monitoring expression of reporter in response to exposure to said compound, relative to expression of reporter in the absence of said compound,

[0075] wherein exposure to said compound is carried out in the presence of:

[0076] a polypeptide comprising at least amino acid residues 461-661 of the protein set forth in SEQ ID NO:2, and

[0077] a reporter construct,

[0078] wherein said reporter construct comprises a reporter gene under the control of a signal dependent transcription factor.

[0079] In accordance with a still further embodiment of the present invention, there are provided methods for treating diabetes mellitus, said method comprising contacting a biological system with an amount of an effective amount of a compound which inhibits binding of CREB to CBP. Such methods ameliorate hyperglycemia associated with diabetes mellitus by modulating gluconeogenesis and/or hyperglucagonemia. Particularly, such methods employ compounds which disrupt the formation of CREB:CBP complexes, thus inhibiting transcription of PEPCK or glucogon gene.

[0080] As employed herein, the phrase “biological system” refers to an intact organism or a cell-based system containing the various components required for response to the ligands described herein, e.g., an isoform of RAR (i.e., RARα, RARβ or RARγ), a silent partner for the RAR isoform (e.g., RXR), and an RAR-responsive reporter (which typically comprises an RAR response element (RARE) in operative communication with a reporter gene; suitable reporters include luciferase, chloramphenicol transferase, β-galactosidase, and the like.

[0081] Contacting in a biological system contemplated by the present invention can be accomplished in a variety of ways, and the treating agents contemplated for use herein can be administered in a variety of forms (e.g., in combination with a pharmaceutically acceptable carrier therefor) and by a variety of modes of delivery. Exemplary pharmaceutically acceptable carriers include carriers suitable for oral, intravenous, subcutaneous, intramuscular, intracutaneous, and the like administration. Administration in the form of creams, lotions, tablets, dispersible powders, granules, syrups, elixirs, sterile aqueous or non-aqueous solutions, suspensions or emulsions, and the like, is contemplated.

[0082] For the preparation of oral liquids, suitable carriers include emulsions, solutions, suspensions, syrups, and the like, optionally containing additives such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents, and the like.

[0083] For the preparation of fluids for parenteral administration, suitable carriers include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile water, or some other sterile injectable medium immediately before use.

[0084] As employed herein, the phrase “effective amount” refers to levels of compound sufficient to provide circulating concentrations high enough to modulate the expression of gene(s) mediated by members of the steroid/thyroid superfamily of receptors. Such a concentration typically falls in the range of about 10 nM up to 2 μM; with concentrations in the range of about 100 nM up to 500 nM being preferred. Since the activity of different compounds described herein may vary considerably, and since individual subjects may present a wide variation in severity of symptoms, it is up to the practitioner to determine a subject's response to treatment and vary the dosages accordingly.

[0085] The invention will now be described in greater detail by reference to the following non-limiting examples.

EXAMPLE I Functional Properties of CBP

[0086] To characterize the functional properties of CBP, rabbit CBP antiserum was developed against a fragment of CBP extending from amino acid residues 634-648 within the CREB binding domain of CBP (i.e., KVEGDMYESANSRDE; SEQ ID NO:3). Crude antiserum was affinity purified on a synthetic CBP peptide column, as described by Gonzalez et al., in Mol. and Cell Biol. 11(3):1306-1312 (1991), which is incorporated herein by reference. Far-Western and Western blot assays were performed as described by, for example, Chrivia et al., in Nature 365:855-859 (1993), also incorporated herein by reference. Thus, Western (CBP) and Far-Western (³²P-CREB) blot analysis of partially purified CBP protein from HeLa nuclear extract was carried out following SDS-PAGE and transfer to nitrocellulose. Far-Western blots were also obtained for crude HeLa nuclear extracts using ³²P-labeled CREB, phosphorylated with PK-A or casein kinase II (CKII). Far-Western blot analysis was also conducted with immunoprecipitates prepared from HeLa nuclear extracts with control IgG or affinity purified CBP antiserum (CBP-Ab). CREB binding activity was detected with ³²P-labeled CREB phosphorylated with PK-A.

[0087] Using the above-described antiserum, a 265 kD polypeptide was detected on Western blots, as predicted from the cDNA (see Chrivia et al., supra), which coincided with the predominant phospho-CREB binding activity in HeLa nuclear extracts by “Far-Western” blot assay. An identical phospho-CREB binding activity was also found in NIH3T3 cells. This phospho-CREB binding protein appeared to be specific for Ser133 phosphorylated CREB because no such band was detected with CREB labeled to the same specific activity at a non-regulatory phospho-acceptor site (Ser156) by casein kinase II (CKII) (see Hagiwara et al., Cell 70:105-113 (1992), which is incorporated herein by reference).

[0088] To further demonstrate that the major phospho-CREB binding protein in HeLa and NIH3T3 cells is specifically bound by the anti-CBP antibody, immunoprecipitates were prepared from crude nuclear extracts using the CBP antiserum. Far-Western analysis of these immunoprecipitates revealed a 265 kD band in samples incubated with CBP antiserum, but not with control IgG.

EXAMPLE II Role of Phosphorylation in CREB-CBP Interaction

[0089] To examine whether the phosphorylation dependent interaction between CREB and CBP was critical for cAMP responsive transcription, a microinjection assay was employed using CBP antiserum, which would be predicted to impair formation of a CREB-CBP complex. Thus, NIH3T3 cells were cultured in 5% CO₂ atmosphere in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal calf serum. Forty-eight hours prior to injection, cells were passaged into scored glass coverslips and made quiescent by incubation in medium containing 0.05% fetal calf serum for 24 hours (see, for example, Hagikara et al., supra and Alberts et al., in Mol. and Cell Biol. 13:2104-2112 (1993), both incorporated herein by reference). Representative fields of NIH3T3 cells were injected with pCRE-lacZ reporter plasmid plus 5, 0.5, and 0.05 mg/ml of affinity purified CBP antiserum. Total antibody concentration in microinjected cells was maintained at 5 mg/ml by adjusting with control Rabbit IgG. Injected cells were stimulated with 0.5 mM 8-bromo-cAMP, plus 3-isobutyl-1-methylxanthine (IBMX) for 4 hours, then fixed and assayed for lacZ activity (β-Gal) as well as antibody content (Texas Red anti-Rb).

[0090] Following microinjection into nuclei of NIH3T3 cells, a CRE-lacZ reporter was markedly induced by treatment with 8-bromo-cAMP plus IBMX. Co-injection of CBP antiserum with the CRE-lacZ plasmid inhibited cAMP dependent activity in a dosage-dependent manner, but control IgG had no effect on this response.

[0091] To determine whether CBP antiserum inhibited cAMP responsive transcription by binding specifically to CBP, peptide blocking experiments were performed. Thus, the effect of CBP antiserum on CRE-lacZ reporter activity following pre-treatment of CBP antiserum with synthetic CBP peptide (anti-CBP+CBP) or unrelated peptide (anti-CBP+ILS; the unrelated peptide, ILS, is described by Leonard et al., in Mol. Endocr. 7: 1275-1283 (1993), which is incorporated herein by reference) was determined. Rabbit IgG+CBP and rabbit IgG pre-treated with CBP peptide were used as controls. NIH3T3 cells were injected with CRE-lacZ reporter plus various CBP antisera, stimulated with 0.5 mM 8-bromo-cAMP, plus IBMX for 4 hours, and assayed for lacZ activity. Cells expressing the lacZ gene product form a blue precipitate upon X-gal staining, which quenches immunofluorescent detection of the injected antibody.

[0092] CBP antiserum, pre-incubated with synthetic CBP peptide, was unable to recognize the 265 kD CBP product on a Western blot, and could not inhibit CRE-lacZ reporter activity upon microinjection into NIH3T3 cells. But antiserum treated with an unrelated synthetic peptide (ILS) retained full activity in both Western and microinjection assay, suggesting that the ability of the antiserum to bind CBP was critical for its inhibitory effect on cAMP dependent transcription.

[0093] Results of these experiments are summarized in FIG. 1.

EXAMPLE III Multiple Signaling Pathways Utilize CBP

[0094] To determine whether CBP activity may be restricted to a subset of promoters, several constitutively active reporter constructs were tested:

[0095] Cytomegalovirus (CMV-lacZ),

[0096] Rous sarcoma virus (RSV-lacZ), and

[0097] SV40 (SV40-lacZ).

[0098] Thus, cells were microinjected with CBP antiserum plus Rous Sarcoma Virus (pRSV-lacZ) or Cytomegalovirus (pCMV-lacZ) reporter constructs. Alternatively, NIH3T3 cells microinjected with CBP antiserum (or non-specific rabbit IgG (RbIgG)), plus reporter constructs containing either cAMP responsive elements (pCRE-lacZ), serum responsive elements (pSRE-lacZ) or phorbol ester responsive elements (pTRE-lacZ). Light field photo-micrographs show cells stained for β-galactosidase activity following four hour treatment with either 0.5 mM 8-bromo-cAMP, plus IBMX (pCRE-lacZ), 20% fetal calf serum (pSRE-lacZ), or 200 ng/ml TPA (pTRE-lacZ). Results of β-galactosidase assays are summarized in FIG. 2. Dark field photos show microinjected IgGs as visualized by immunofluorescence using Texas Red donkey anti-rabbit IgG.

[0099] When examined in NIH3T3 cells by transient transfection assay, each of the constitutively active reporter constructs had comparable basal activity, relative to the cAMP-stimulated CRE reporter plasmid, thereby permitting the effects of CBP antiserum on these reporters to be compared directly. Although co-injected CBP antiserum could block cAMP stimulated activity from a CRE-lacZ reporter in contemporaneous assays, no inhibition was observed on basal expression from any of the constitutive promoter constructs tested, even when 10-fold lower amounts of reporter plasmid were employed.

[0100] These results suggest that CBP can indeed discriminate between basal and signal dependent activities in vivo.

EXAMPLE IV CBP-Involvement in Non-CREB Mediated Pathways

[0101] Previous reports showing that serum and phorbol esters stimulate their target genes through phosphorylation-dependent trans-activators (see, for example, Hill et al., in Cell 73:395-406 (1993) or Smeal et al., in Nature 354:494-496 (1991), both incorporated herein by reference), suggested that CBP might also function in these signaling pathways. Thus, Far-Western analyses were carried out with crude HeLa nuclear extracts using ³²P-labeled recombinant Jun protein phosphorylated in vitro with either Jun-kinase (JNK; see Hibi et al., in Genes and Develop. 7:2135-2148 (1993), incorporated herein by reference) or casein kinase II (CK II).

[0102] Whereas serum and TPA could stimulate reporter activity in NIH3T3 cells microinjected with serum responsive element (SRE)-lacZ and TPA-responsive element (TRE)-lacZ indicator plasmids, respectively, co-injected CBP antiserum completely blocked both responses. These results suggest that CBP not only interacts with CREB, but also with other signal-dependent transcription factors.

[0103] In this regard, phorbol esters and serum induce TRE-dependent transcription, in part, through the Jun-kinase (JNK) mediated phosphorylation of c-Jun at Ser63 and Ser73 (see, for example, Smeal et al., supra or Hibi et al., supra). Using ³²P-labeled recombinant c-Jun protein, phosphorylated at Ser63 and Ser73 with JNK, Far-Western blot assays were performed on crude HeLa nuclear extracts. JNK-phosphorylated c-Jun protein could bind CBP with comparable affinity to CREB. But c-Jun labeled to similar specific activity at non-activating sites (Thr 231, Ser243, and Ser249; see Boyle et al., in Cell 64:573-584 (1991)) by CKII, could not interact with CBP, suggesting that interaction between CBP and c-Jun requires phosphorylation of the transcriptionally active Ser63 and Ser73 phospho-acceptor sites. In view of the inhibitory effect of CBP antiserum on TRE-β gal reporter expression following phorbol ester and serum induction, the phosphorylation dependent interaction between CBP and c-Jun would appear to be a critical component of these response pathways.

EXAMPLE V Chromatographic Purification of CBP

[0104] Based on the surprising discovery that CBP cooperates with phosphorylation dependent activators by recruiting general transcription factors to target promoters, it was next examined whether CBP would co-fractionate with any general factors in HeLa nuclear extracts. Thus, Far-Western analyses of protein fractions were obtained after phospho-cellulose chromatography. Phospho-CREB binding proteins were visualized using ³²P-labeled CREB phosphorylated in vitro with PK-A (³²P-CREB). Western analysis was carried out with the same blot as described above, using affinity purified CBP antibody (CBP Ab). Far-Western (³²P-CREB) and. Western (CBP-Ab) analyses of fractions were also carried out following DEAE and DE52 chromatography. Phosphocellulose, DEAE, and DE52 chromatography was performed on HeLa nuclear extracts as described by Ferreri et al., in Proc. Natl. Acad. Sci. USA in press (1993), which is incorporated herein by reference.

[0105] Both CBP-immunoreactive and phospho-CREB binding activities were retained on phosphocellulose columns and were eluted at 0.3-0.5M KCl. Further purification of a comparable phospho-cellulose fraction on DEAE-sepharose and DE52 resins showed that CBP was highly enriched in fractions containing TFII (E, F, H) but not TFIID activities. Although the general factor which associates directly with CBP is not known, the co-fractionation of CBP with proteins involved in basal transcription initiation suggests a testable mechanism for CBP action. In particular, the results presented herein suggest that phosphorylation-dependent activators like CREB and Jun influence assembly of late-acting factors (TFII E, F, H) during transcriptional initiation/reinitiation by interacting with CBP in a signal dependent manner.

[0106] While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

1 4 1 7326 DNA Mus CDS (1)...(7326) misc_feature (1)...(7326) n = A,T,C or G 1 atg gcc gag aac ttg ctg gac gga ccg ccc aac ccc aaa cga gcc aaa 48 Met Ala Glu Asn Leu Leu Asp Gly Pro Pro Asn Pro Lys Arg Ala Lys 1 5 10 15 ctc agc tcg ccc ggc ttc tcc gcg aat gac aac aca gat ttt gga tca 96 Leu Ser Ser Pro Gly Phe Ser Ala Asn Asp Asn Thr Asp Phe Gly Ser 20 25 30 ttg ttt gac ttg gaa aat gac ctt cct gat gag ctg atc ccc aat gga 144 Leu Phe Asp Leu Glu Asn Asp Leu Pro Asp Glu Leu Ile Pro Asn Gly 35 40 45 gaa tta agc ctt tta aac agt ggg aac ctt gtt cca gat gct gcg tcc 192 Glu Leu Ser Leu Leu Asn Ser Gly Asn Leu Val Pro Asp Ala Ala Ser 50 55 60 aaa cat aaa caa ctg tca gag ctt ctt aga gga ggc agc ggc tct agc 240 Lys His Lys Gln Leu Ser Glu Leu Leu Arg Gly Gly Ser Gly Ser Ser 65 70 75 80 atc aac cca ggg ata ggc aat gtg agt gcc agc agc cct gtg caa cag 288 Ile Asn Pro Gly Ile Gly Asn Val Ser Ala Ser Ser Pro Val Gln Gln 85 90 95 ggc ctt ggt ggc cag gct cag ggg cag ccg aac agt aca aac atg gcc 336 Gly Leu Gly Gly Gln Ala Gln Gly Gln Pro Asn Ser Thr Asn Met Ala 100 105 110 agc tta ggt gcc atg ggc aag agc cct ctg aac caa gga gac tca tca 384 Ser Leu Gly Ala Met Gly Lys Ser Pro Leu Asn Gln Gly Asp Ser Ser 115 120 125 aca ccc aac ctg ccc aaa cag gca gcc agc acc tct ggg ccc act ccc 432 Thr Pro Asn Leu Pro Lys Gln Ala Ala Ser Thr Ser Gly Pro Thr Pro 130 135 140 cct gcc tcc caa gca ctg aat cca caa gca caa aag caa gta ggg ctg 480 Pro Ala Ser Gln Ala Leu Asn Pro Gln Ala Gln Lys Gln Val Gly Leu 145 150 155 160 gtg acc agt agt cct gcc aca tca cag act gga cct ggg atc tgc atg 528 Val Thr Ser Ser Pro Ala Thr Ser Gln Thr Gly Pro Gly Ile Cys Met 165 170 175 aat gct aac ttc aac cag acc cac cca ggc ctt ctc aat agt aac tct 576 Asn Ala Asn Phe Asn Gln Thr His Pro Gly Leu Leu Asn Ser Asn Ser 180 185 190 ggc cat agc tta atg aat cag gct caa caa ggg caa gct caa gtc atg 624 Gly His Ser Leu Met Asn Gln Ala Gln Gln Gly Gln Ala Gln Val Met 195 200 205 aat gga tct ctt ggg gct gct gga aga gga agg gga gct gga atg ccc 672 Asn Gly Ser Leu Gly Ala Ala Gly Arg Gly Arg Gly Ala Gly Met Pro 210 215 220 tac cct gct cca gcc atg cag ggg gcc aca agc agt gtg ctg gcg gag 720 Tyr Pro Ala Pro Ala Met Gln Gly Ala Thr Ser Ser Val Leu Ala Glu 225 230 235 240 acc ttg aca cag gtt tcc cca caa atg gct ggc cat gct gga cta aat 768 Thr Leu Thr Gln Val Ser Pro Gln Met Ala Gly His Ala Gly Leu Asn 245 250 255 aca gca cag gca gga ggc atg acc aag atg gga atg act ggt acc aca 816 Thr Ala Gln Ala Gly Gly Met Thr Lys Met Gly Met Thr Gly Thr Thr 260 265 270 agt cca ttt gga caa ccc ttt agt caa act gga ggg cag cag atg gga 864 Ser Pro Phe Gly Gln Pro Phe Ser Gln Thr Gly Gly Gln Gln Met Gly 275 280 285 gcc act gga gtg aac ccc cag tta gcc agc aaa cag agc atg gtc aat 912 Ala Thr Gly Val Asn Pro Gln Leu Ala Ser Lys Gln Ser Met Val Asn 290 295 300 agt tta cct gct ttt cct aca gat atc aag aat act tca gtc acc act 960 Ser Leu Pro Ala Phe Pro Thr Asp Ile Lys Asn Thr Ser Val Thr Thr 305 310 315 320 gtg cca aat atg tcc cag ttg caa aca tca gtg gga att gta ccc aca 1008 Val Pro Asn Met Ser Gln Leu Gln Thr Ser Val Gly Ile Val Pro Thr 325 330 335 caa gca att gca aca ggc ccc aca gca gac cct gaa aaa cgc aaa ctg 1056 Gln Ala Ile Ala Thr Gly Pro Thr Ala Asp Pro Glu Lys Arg Lys Leu 340 345 350 ata cag cag cag ctg gtt cta ctg ctt cat gcc cac aaa tgt cag aga 1104 Ile Gln Gln Gln Leu Val Leu Leu Leu His Ala His Lys Cys Gln Arg 355 360 365 cga gag caa gca aat gga gag gtt cgn gcc tgt tct ctc cca cac tgt 1152 Arg Glu Gln Ala Asn Gly Glu Val Xaa Ala Cys Ser Leu Pro His Cys 370 375 380 cga acc atg aaa aac gtt ttg aat cac atg aca cat tgt cag gct ccc 1200 Arg Thr Met Lys Asn Val Leu Asn His Met Thr His Cys Gln Ala Pro 385 390 395 400 aaa gcc tgc caa gtt gcc cat tgt gca tct tca cga caa atc atc tct 1248 Lys Ala Cys Gln Val Ala His Cys Ala Ser Ser Arg Gln Ile Ile Ser 405 410 415 cat tgg aag aac tgc aca cga cat gac tgt cct gtt tgc ctc cct ttg 1296 His Trp Lys Asn Cys Thr Arg His Asp Cys Pro Val Cys Leu Pro Leu 420 425 430 aaa aat gcc agt gac aag cga aac caa caa acc atc ctg gga tct cca 1344 Lys Asn Ala Ser Asp Lys Arg Asn Gln Gln Thr Ile Leu Gly Ser Pro 435 440 445 gct agt gga att caa aac aca att ggt tct gtt ggt gca ggg caa cag 1392 Ala Ser Gly Ile Gln Asn Thr Ile Gly Ser Val Gly Ala Gly Gln Gln 450 455 460 aat gcc act tcc tta agt aac cca aat ccc ata gac ccc agt tcc atg 1440 Asn Ala Thr Ser Leu Ser Asn Pro Asn Pro Ile Asp Pro Ser Ser Met 465 470 475 480 cag cgg gcc tat gct gct cta gga ctc ccc tac atg aac cag cct cag 1488 Gln Arg Ala Tyr Ala Ala Leu Gly Leu Pro Tyr Met Asn Gln Pro Gln 485 490 495 acg cag ctg cag cct cag gtt cct ggc cag caa cca gca cag cct cca 1536 Thr Gln Leu Gln Pro Gln Val Pro Gly Gln Gln Pro Ala Gln Pro Pro 500 505 510 gcc cac cag cag atg agg act ctc aat gcc cta gga aac aac ccc atg 1584 Ala His Gln Gln Met Arg Thr Leu Asn Ala Leu Gly Asn Asn Pro Met 515 520 525 agt gtc cca gca gga gga ata aca aca gat caa cag cca cca aac ttg 1632 Ser Val Pro Ala Gly Gly Ile Thr Thr Asp Gln Gln Pro Pro Asn Leu 530 535 540 att tca gaa tca gct ctt cca act tcc ttg ggg gct acc aat cca ctg 1680 Ile Ser Glu Ser Ala Leu Pro Thr Ser Leu Gly Ala Thr Asn Pro Leu 545 550 555 560 atg aat gat ggt tca aac tct ggt aac att gga agc ctc agc acg ata 1728 Met Asn Asp Gly Ser Asn Ser Gly Asn Ile Gly Ser Leu Ser Thr Ile 565 570 575 cct aca gca gcg cct cct tcc agc act ggt gtt cga aaa ggc tgg cat 1776 Pro Thr Ala Ala Pro Pro Ser Ser Thr Gly Val Arg Lys Gly Trp His 580 585 590 gaa cat gtg act cag gac cta cgg agt cat cta gtc cat aaa ctc gtt 1824 Glu His Val Thr Gln Asp Leu Arg Ser His Leu Val His Lys Leu Val 595 600 605 caa gcc atc ttc cca act cca gac cct gca gct ctg aaa gat cgc cgc 1872 Gln Ala Ile Phe Pro Thr Pro Asp Pro Ala Ala Leu Lys Asp Arg Arg 610 615 620 atg gag aac ctg gtt gcc tat gct aag aaa gtg gag gga gac atg tat 1920 Met Glu Asn Leu Val Ala Tyr Ala Lys Lys Val Glu Gly Asp Met Tyr 625 630 635 640 gag tct gct aat agc agg gat gaa tac tat cat tta tta gca gag aaa 1968 Glu Ser Ala Asn Ser Arg Asp Glu Tyr Tyr His Leu Leu Ala Glu Lys 645 650 655 atc tat aaa ata caa aaa gaa cta gaa gaa aag cgg agg aca cgt tta 2016 Ile Tyr Lys Ile Gln Lys Glu Leu Glu Glu Lys Arg Arg Thr Arg Leu 660 665 670 cat aag caa ggc atc ctg ggt aac cag cca gct tta cca gct tct ggg 2064 His Lys Gln Gly Ile Leu Gly Asn Gln Pro Ala Leu Pro Ala Ser Gly 675 680 685 gct cag ccc cct gtg att cca cca gcc cag tct gta aga cct cca aat 2112 Ala Gln Pro Pro Val Ile Pro Pro Ala Gln Ser Val Arg Pro Pro Asn 690 695 700 ggg ccc ctg cct ttg cca gtg aat cgc atg cag gtt tct caa ggg atg 2160 Gly Pro Leu Pro Leu Pro Val Asn Arg Met Gln Val Ser Gln Gly Met 705 710 715 720 aat tca ttt aac cca atg tcc ctg gga aac gtc cag ttg cca cag gca 2208 Asn Ser Phe Asn Pro Met Ser Leu Gly Asn Val Gln Leu Pro Gln Ala 725 730 735 ccc atg gga cct cgt gca gcc tcc cct atg aac cac tct gtg cag atg 2256 Pro Met Gly Pro Arg Ala Ala Ser Pro Met Asn His Ser Val Gln Met 740 745 750 aac agc atg gcc tca gtt ccg ggt atg gcc att tct cct tca cgg atg 2304 Asn Ser Met Ala Ser Val Pro Gly Met Ala Ile Ser Pro Ser Arg Met 755 760 765 cct cag cct cca aat atg atg ggc act cat gcc aac aac att atg gcc 2352 Pro Gln Pro Pro Asn Met Met Gly Thr His Ala Asn Asn Ile Met Ala 770 775 780 cag gca cct act cag aac cag ttt ctg cca cag aac cag ttt cca tca 2400 Gln Ala Pro Thr Gln Asn Gln Phe Leu Pro Gln Asn Gln Phe Pro Ser 785 790 795 800 tcc agt ggg gca atg agt gtg aac agt gtg ggc atg ggg caa cca gca 2448 Ser Ser Gly Ala Met Ser Val Asn Ser Val Gly Met Gly Gln Pro Ala 805 810 815 gcc cag gca ggt gtt tca cag ggt cag gaa cct gga gct gct ctc cct 2496 Ala Gln Ala Gly Val Ser Gln Gly Gln Glu Pro Gly Ala Ala Leu Pro 820 825 830 aac cct ctg aac atg ctg gca ccc cag gcc agc cag ctg cct tgc cca 2544 Asn Pro Leu Asn Met Leu Ala Pro Gln Ala Ser Gln Leu Pro Cys Pro 835 840 845 cca gtg aca cag tca cca ttg cac ccg act cca cct cct gct tcc aca 2592 Pro Val Thr Gln Ser Pro Leu His Pro Thr Pro Pro Pro Ala Ser Thr 850 855 860 gct gct ggc atg ccc tct ctc caa cat cca acg gca cca gga atg acc 2640 Ala Ala Gly Met Pro Ser Leu Gln His Pro Thr Ala Pro Gly Met Thr 865 870 875 880 cct cct cag cca gca gct ccc act cag cca tct act cct gtg tca tct 2688 Pro Pro Gln Pro Ala Ala Pro Thr Gln Pro Ser Thr Pro Val Ser Ser 885 890 895 ggg cag act cct acc cca act cct ggc tca gtg ccc agc gct gcc caa 2736 Gly Gln Thr Pro Thr Pro Thr Pro Gly Ser Val Pro Ser Ala Ala Gln 900 905 910 aca cag agt acc cct aca gtc cag gca gca gca cag gct cag gtg act 2784 Thr Gln Ser Thr Pro Thr Val Gln Ala Ala Ala Gln Ala Gln Val Thr 915 920 925 cca cag cct cag acc cca gtg cag cca cca tct gtg gct act cct cag 2832 Pro Gln Pro Gln Thr Pro Val Gln Pro Pro Ser Val Ala Thr Pro Gln 930 935 940 tca tca cag cag caa cca acg cct gtg cat act cag cca cct ggc aca 2880 Ser Ser Gln Gln Gln Pro Thr Pro Val His Thr Gln Pro Pro Gly Thr 945 950 955 960 ccg ctt tct cag gca gca gcc agc att gat aat aga gtc cct act ccc 2928 Pro Leu Ser Gln Ala Ala Ala Ser Ile Asp Asn Arg Val Pro Thr Pro 965 970 975 tcc act gtg acc agt gct gaa acc agt tcc cag cag cca gga ccc gat 2976 Ser Thr Val Thr Ser Ala Glu Thr Ser Ser Gln Gln Pro Gly Pro Asp 980 985 990 gtg ccc atg ctg gaa atg aag aca gag gtg cag aca gat gat gct gag 3024 Val Pro Met Leu Glu Met Lys Thr Glu Val Gln Thr Asp Asp Ala Glu 995 1000 1005 cct gaa cct act gaa tcc aag ggg gaa cct cgg tct gag atg atg gaa 3072 Pro Glu Pro Thr Glu Ser Lys Gly Glu Pro Arg Ser Glu Met Met Glu 1010 1015 1020 gag gat tta caa ggt tct tcc caa gta aaa gaa gag aca gat acg aca 3120 Glu Asp Leu Gln Gly Ser Ser Gln Val Lys Glu Glu Thr Asp Thr Thr 1025 1030 1035 1040 gag cag aag tca gag cca atg gaa gta gaa gaa aag aaa cct gaa gta 3168 Glu Gln Lys Ser Glu Pro Met Glu Val Glu Glu Lys Lys Pro Glu Val 1045 1050 1055 aaa gtg gaa gct aaa gag gaa gaa gag aac agt tcg aac gac aca gcc 3216 Lys Val Glu Ala Lys Glu Glu Glu Glu Asn Ser Ser Asn Asp Thr Ala 1060 1065 1070 tca caa tca aca tct cct tcc cag cca cgc aaa aaa atc ttt aaa ccc 3264 Ser Gln Ser Thr Ser Pro Ser Gln Pro Arg Lys Lys Ile Phe Lys Pro 1075 1080 1085 gag gag cta cgc cag gca ctt atg cca act cta gaa gca ctc tat cga 3312 Glu Glu Leu Arg Gln Ala Leu Met Pro Thr Leu Glu Ala Leu Tyr Arg 1090 1095 1100 cag gac cca gag tct ttg cct ttt cgt cag cct gta gat cct cag ctc 3360 Gln Asp Pro Glu Ser Leu Pro Phe Arg Gln Pro Val Asp Pro Gln Leu 1105 1110 1115 1120 cta gga atc cca gat tat ttt gat ata gtg aag aat cct atg gac ctt 3408 Leu Gly Ile Pro Asp Tyr Phe Asp Ile Val Lys Asn Pro Met Asp Leu 1125 1130 1135 tct acc atc aaa cga aag ctg gac aca ggg caa tat caa gaa ccc tgg 3456 Ser Thr Ile Lys Arg Lys Leu Asp Thr Gly Gln Tyr Gln Glu Pro Trp 1140 1145 1150 cag tat gtg gat gat gtc agg ctt atg ttc aac aat gcg tgg cta tat 3504 Gln Tyr Val Asp Asp Val Arg Leu Met Phe Asn Asn Ala Trp Leu Tyr 1155 1160 1165 aat cgt aaa acg tcc cgt gta tat aaa ttt tgc agt aaa ctt gca gag 3552 Asn Arg Lys Thr Ser Arg Val Tyr Lys Phe Cys Ser Lys Leu Ala Glu 1170 1175 1180 gtc ttt gaa caa gaa att gac cct gtc atg cag tct ctt gga tat tgc 3600 Val Phe Glu Gln Glu Ile Asp Pro Val Met Gln Ser Leu Gly Tyr Cys 1185 1190 1195 1200 tgt gga cga aag tat gag ttc tcc cca cag act ttg tgc tgt tac gga 3648 Cys Gly Arg Lys Tyr Glu Phe Ser Pro Gln Thr Leu Cys Cys Tyr Gly 1205 1210 1215 aag cag ctg tgt aca att cct cgt gat gca gcc tac tac agc tat cag 3696 Lys Gln Leu Cys Thr Ile Pro Arg Asp Ala Ala Tyr Tyr Ser Tyr Gln 1220 1225 1230 aat agg tat cat ttc tgt ggg aag tgt ttc aca gag atc cag ggc gag 3744 Asn Arg Tyr His Phe Cys Gly Lys Cys Phe Thr Glu Ile Gln Gly Glu 1235 1240 1245 aat gtg acc ctg ggt gac gac cct tcc caa cct cag acg aca att tcc 3792 Asn Val Thr Leu Gly Asp Asp Pro Ser Gln Pro Gln Thr Thr Ile Ser 1250 1255 1260 aag gat caa ttt gaa aag aag aaa aat gat acc tta gat cct gaa cct 3840 Lys Asp Gln Phe Glu Lys Lys Lys Asn Asp Thr Leu Asp Pro Glu Pro 1265 1270 1275 1280 ttt gtt gac tgc aaa gag tgt ggc cgg aag atg cat cag att tgt gtt 3888 Phe Val Asp Cys Lys Glu Cys Gly Arg Lys Met His Gln Ile Cys Val 1285 1290 1295 cta cac tat gac atc att tgg cct tca ggt ttt gtg tgt gac aac tgt 3936 Leu His Tyr Asp Ile Ile Trp Pro Ser Gly Phe Val Cys Asp Asn Cys 1300 1305 1310 ttg aag aaa act ggc aga cct cgg aaa gaa aac aaa ttc agt gct aag 3984 Leu Lys Lys Thr Gly Arg Pro Arg Lys Glu Asn Lys Phe Ser Ala Lys 1315 1320 1325 agg ctg cag acc aca cga ttg gga aac cac tta gaa gac aga gtg aat 4032 Arg Leu Gln Thr Thr Arg Leu Gly Asn His Leu Glu Asp Arg Val Asn 1330 1335 1340 aag ttt ttg cgg cgc cag aat cac cct gaa gct ggg gag gtt ttt gtc 4080 Lys Phe Leu Arg Arg Gln Asn His Pro Glu Ala Gly Glu Val Phe Val 1345 1350 1355 1360 aga gtg gtg gcc agc tca gac aag act gtg gag gtc aag ccg gga atg 4128 Arg Val Val Ala Ser Ser Asp Lys Thr Val Glu Val Lys Pro Gly Met 1365 1370 1375 aag tca agg ttt gtg gat tct gga gag atg tcg gaa tct ttc cca tat 4176 Lys Ser Arg Phe Val Asp Ser Gly Glu Met Ser Glu Ser Phe Pro Tyr 1380 1385 1390 cgt acc aaa gca ctc ttt gct ttt gag gag atc gat gga gtc gat gtg 4224 Arg Thr Lys Ala Leu Phe Ala Phe Glu Glu Ile Asp Gly Val Asp Val 1395 1400 1405 tgc ttt ttt ggg atg cat gtg caa gat acg gct ctg att gcc ccc cac 4272 Cys Phe Phe Gly Met His Val Gln Asp Thr Ala Leu Ile Ala Pro His 1410 1415 1420 caa ata caa ggc tgt gta tac ata tct tat ctg gac agt att cat ttc 4320 Gln Ile Gln Gly Cys Val Tyr Ile Ser Tyr Leu Asp Ser Ile His Phe 1425 1430 1435 1440 ttc cgg ccc cgc tgc ctc cgg aca gct gtt tac cat gag atc ctc atc 4368 Phe Arg Pro Arg Cys Leu Arg Thr Ala Val Tyr His Glu Ile Leu Ile 1445 1450 1455 gga tat ctc gag tat gtg aag aaa ttg gtg tat gtg aca gca cat att 4416 Gly Tyr Leu Glu Tyr Val Lys Lys Leu Val Tyr Val Thr Ala His Ile 1460 1465 1470 tgg gcc tgt ccc cca agt gaa gga gat gac tat atc ttt cat tgc cac 4464 Trp Ala Cys Pro Pro Ser Glu Gly Asp Asp Tyr Ile Phe His Cys His 1475 1480 1485 ccc cct gac cag aaa atc ccc aaa cca aaa cga cta cag gag tgg tac 4512 Pro Pro Asp Gln Lys Ile Pro Lys Pro Lys Arg Leu Gln Glu Trp Tyr 1490 1495 1500 aag aag atg ctg gac aag gcg ttt gca gag agg atc att aac gac tat 4560 Lys Lys Met Leu Asp Lys Ala Phe Ala Glu Arg Ile Ile Asn Asp Tyr 1505 1510 1515 1520 aag gac atc ttc aaa caa gcg aac gaa gac agg ctc acg agt gcc aag 4608 Lys Asp Ile Phe Lys Gln Ala Asn Glu Asp Arg Leu Thr Ser Ala Lys 1525 1530 1535 gag ttg ccc tat ttt gaa gga gat ttc tgg cct aat gtg ttg gaa gaa 4656 Glu Leu Pro Tyr Phe Glu Gly Asp Phe Trp Pro Asn Val Leu Glu Glu 1540 1545 1550 agc att aag gaa cta gaa caa gaa gaa gaa gaa agg aaa aaa gaa gag 4704 Ser Ile Lys Glu Leu Glu Gln Glu Glu Glu Glu Arg Lys Lys Glu Glu 1555 1560 1565 agt act gca gcg agt gag act cct gag ggc agt cag ggt gac agc aaa 4752 Ser Thr Ala Ala Ser Glu Thr Pro Glu Gly Ser Gln Gly Asp Ser Lys 1570 1575 1580 aat gcg aag aaa aag aac aac aag aag acc aac aaa aac aaa agc agc 4800 Asn Ala Lys Lys Lys Asn Asn Lys Lys Thr Asn Lys Asn Lys Ser Ser 1585 1590 1595 1600 att agc cgc gcc aac aag aag aag ccc agc atg ccc aat gtt tcc aac 4848 Ile Ser Arg Ala Asn Lys Lys Lys Pro Ser Met Pro Asn Val Ser Asn 1605 1610 1615 gac ctg tcg cag aag ctg tat gcc acc atg gag aag cac aag gag gta 4896 Asp Leu Ser Gln Lys Leu Tyr Ala Thr Met Glu Lys His Lys Glu Val 1620 1625 1630 ttc ttt gtg att cat ctg cat gct ggg cct gtt atc agc act cag ccc 4944 Phe Phe Val Ile His Leu His Ala Gly Pro Val Ile Ser Thr Gln Pro 1635 1640 1645 ccc atc gtg gac cct gat cct ctg ctt agc tgt gac ctc atg gat ggg 4992 Pro Ile Val Asp Pro Asp Pro Leu Leu Ser Cys Asp Leu Met Asp Gly 1650 1655 1660 cga gat gcc ttc ctc acc ctg gcc aga gac aag cac tgg gaa ttc tct 5040 Arg Asp Ala Phe Leu Thr Leu Ala Arg Asp Lys His Trp Glu Phe Ser 1665 1670 1675 1680 tcc tta cgc cgc tcc aaa tgg tcc act ctg tgc atg ctg gtg gag ctg 5088 Ser Leu Arg Arg Ser Lys Trp Ser Thr Leu Cys Met Leu Val Glu Leu 1685 1690 1695 cac aca cag ggc cag gac cgc ttt gtt tat acc tgc aat gag tgc aaa 5136 His Thr Gln Gly Gln Asp Arg Phe Val Tyr Thr Cys Asn Glu Cys Lys 1700 1705 1710 cac cat gtg gaa aca cgc tgg cac tgc act gtg tgt gag gac tat gac 5184 His His Val Glu Thr Arg Trp His Cys Thr Val Cys Glu Asp Tyr Asp 1715 1720 1725 ctt tgt atc aat tgc tac aac aca aag agc cac acc cat aag atg gtg 5232 Leu Cys Ile Asn Cys Tyr Asn Thr Lys Ser His Thr His Lys Met Val 1730 1735 1740 aag tgg ggg cta ggc cta gat gat gag ggc agc agt cag ggt gag cca 5280 Lys Trp Gly Leu Gly Leu Asp Asp Glu Gly Ser Ser Gln Gly Glu Pro 1745 1750 1755 1760 cag tcc aag agc ccc cag gaa tcc cgg cgt ctc agc atc cag cgc tgc 5328 Gln Ser Lys Ser Pro Gln Glu Ser Arg Arg Leu Ser Ile Gln Arg Cys 1765 1770 1775 atc cag tcc ctg gtg cat gcc tgc cag tgt cgc aat gcc aac tgc tca 5376 Ile Gln Ser Leu Val His Ala Cys Gln Cys Arg Asn Ala Asn Cys Ser 1780 1785 1790 ctg ccg tct tgc cag aag atg aag cga gtc gtg cag cac acc aag ggc 5424 Leu Pro Ser Cys Gln Lys Met Lys Arg Val Val Gln His Thr Lys Gly 1795 1800 1805 tgc aag cgc aag act aat gga gga tgc cca gtg tgc aag cag ctc att 5472 Cys Lys Arg Lys Thr Asn Gly Gly Cys Pro Val Cys Lys Gln Leu Ile 1810 1815 1820 gct ctt tgc tgc tac cac gcc aaa cac tgc caa gaa aat aaa tgc cct 5520 Ala Leu Cys Cys Tyr His Ala Lys His Cys Gln Glu Asn Lys Cys Pro 1825 1830 1835 1840 gtg ccc ttc tgc ctc aac atc aaa cat aac gtc cgc cag cag cag atc 5568 Val Pro Phe Cys Leu Asn Ile Lys His Asn Val Arg Gln Gln Gln Ile 1845 1850 1855 cag cac tgc ctg cag cag gct cag ctc atg cgc cgg cga atg gca acc 5616 Gln His Cys Leu Gln Gln Ala Gln Leu Met Arg Arg Arg Met Ala Thr 1860 1865 1870 atg aac acc cgc aat gtg cct cag cag agt ttg cct tct cct acc tca 5664 Met Asn Thr Arg Asn Val Pro Gln Gln Ser Leu Pro Ser Pro Thr Ser 1875 1880 1885 gca cca ccc ggg act cct aca cag cag ccc agc aca ccc caa aca cca 5712 Ala Pro Pro Gly Thr Pro Thr Gln Gln Pro Ser Thr Pro Gln Thr Pro 1890 1895 1900 cag ccc cca gcc cag cct cag cct tca cct gtt aac atg tca cca gca 5760 Gln Pro Pro Ala Gln Pro Gln Pro Ser Pro Val Asn Met Ser Pro Ala 1905 1910 1915 1920 ggc ttc cct aat gta gcc cgg act cag ccc cca aca ata gtg tct gct 5808 Gly Phe Pro Asn Val Ala Arg Thr Gln Pro Pro Thr Ile Val Ser Ala 1925 1930 1935 ggg aag cct acc aac cag gtg cca gct ccc cca ccc cct gcc cag ccc 5856 Gly Lys Pro Thr Asn Gln Val Pro Ala Pro Pro Pro Pro Ala Gln Pro 1940 1945 1950 cca cct gca gca gta gaa gca gcc cgg caa att gaa cgt gag gcc cag 5904 Pro Pro Ala Ala Val Glu Ala Ala Arg Gln Ile Glu Arg Glu Ala Gln 1955 1960 1965 cag cag cag cac cta tac cga gca aac atc aac aat ggc atg ccc cca 5952 Gln Gln Gln His Leu Tyr Arg Ala Asn Ile Asn Asn Gly Met Pro Pro 1970 1975 1980 gga cgt gac ggt atg ggg acc cca gga agc caa atg act cct gtg ggc 6000 Gly Arg Asp Gly Met Gly Thr Pro Gly Ser Gln Met Thr Pro Val Gly 1985 1990 1995 2000 ctg aat gtg ccc cgt ccc aac caa gtc agt ggg cct gtc atg tct agt 6048 Leu Asn Val Pro Arg Pro Asn Gln Val Ser Gly Pro Val Met Ser Ser 2005 2010 2015 atg cca cct ggg cag tgg cag cag gca ccc atc cct cag cag cag ccg 6096 Met Pro Pro Gly Gln Trp Gln Gln Ala Pro Ile Pro Gln Gln Gln Pro 2020 2025 2030 atg cca ggc atg ccc agg cct gta atg tcc atg cag gcc cag gca gca 6144 Met Pro Gly Met Pro Arg Pro Val Met Ser Met Gln Ala Gln Ala Ala 2035 2040 2045 gtg gct ggg cca cgg atg ccc aat gtg cag cca aac agg agc atc tcg 6192 Val Ala Gly Pro Arg Met Pro Asn Val Gln Pro Asn Arg Ser Ile Ser 2050 2055 2060 cca agt gcc ctg caa gac ctg cta cgg acc cta aag tca ccc agc tct 6240 Pro Ser Ala Leu Gln Asp Leu Leu Arg Thr Leu Lys Ser Pro Ser Ser 2065 2070 2075 2080 cct cag cag cag cag cag gtg ctg aac atc ctt aaa tca aac cca cag 6288 Pro Gln Gln Gln Gln Gln Val Leu Asn Ile Leu Lys Ser Asn Pro Gln 2085 2090 2095 cta atg gca gct ttc atc aaa cag cgc aca gcc aag tat gtg gcc aat 6336 Leu Met Ala Ala Phe Ile Lys Gln Arg Thr Ala Lys Tyr Val Ala Asn 2100 2105 2110 cag cct ggc atg cag ccc cag ccc gga ctt caa tcc cag cct ggt atg 6384 Gln Pro Gly Met Gln Pro Gln Pro Gly Leu Gln Ser Gln Pro Gly Met 2115 2120 2125 cag ccc cag cct ggc atg cac cag cag cct agt ttg caa aac ctg aac 6432 Gln Pro Gln Pro Gly Met His Gln Gln Pro Ser Leu Gln Asn Leu Asn 2130 2135 2140 gca atg caa gct ggt gtg cca cgg cct ggt gtg cct cca cca caa cca 6480 Ala Met Gln Ala Gly Val Pro Arg Pro Gly Val Pro Pro Pro Gln Pro 2145 2150 2155 2160 gca atg gga ggc ctg aat ccc cag gga caa gct ctg aac atc atg aac 6528 Ala Met Gly Gly Leu Asn Pro Gln Gly Gln Ala Leu Asn Ile Met Asn 2165 2170 2175 cca gga cac aac ccc aac atg aca aac atg aat cca cag tac cga gaa 6576 Pro Gly His Asn Pro Asn Met Thr Asn Met Asn Pro Gln Tyr Arg Glu 2180 2185 2190 atg gtg agg aga cag ctg cta cag cac cag cag cag cag cag caa cag 6624 Met Val Arg Arg Gln Leu Leu Gln His Gln Gln Gln Gln Gln Gln Gln 2195 2200 2205 cag cag cag cag cag caa caa caa aat agt gcc agc ttg gcc ggg ggc 6672 Gln Gln Gln Gln Gln Gln Gln Gln Asn Ser Ala Ser Leu Ala Gly Gly 2210 2215 2220 atg gcg gga cac agc cag ttc cag cag cca caa gga cct gga ggt tat 6720 Met Ala Gly His Ser Gln Phe Gln Gln Pro Gln Gly Pro Gly Gly Tyr 2225 2230 2235 2240 gcc cca gcc atg cag cag caa cgc atg caa cag cac ctc ccc atc cag 6768 Ala Pro Ala Met Gln Gln Gln Arg Met Gln Gln His Leu Pro Ile Gln 2245 2250 2255 ggc agc tcc atg ggc cag atg gct gct cca atg gga caa ctt ggc cag 6816 Gly Ser Ser Met Gly Gln Met Ala Ala Pro Met Gly Gln Leu Gly Gln 2260 2265 2270 atg ggg cag cct ggg cta ggg gca gac agc acc cct aat atc cag cag 6864 Met Gly Gln Pro Gly Leu Gly Ala Asp Ser Thr Pro Asn Ile Gln Gln 2275 2280 2285 gcc ctg cag caa cgg att ctg cag cag cag cag atg aag caa caa att 6912 Ala Leu Gln Gln Arg Ile Leu Gln Gln Gln Gln Met Lys Gln Gln Ile 2290 2295 2300 ggg tca cca ggc cag ccg aac ccc atg agc ccc cag cag cac atg ctc 6960 Gly Ser Pro Gly Gln Pro Asn Pro Met Ser Pro Gln Gln His Met Leu 2305 2310 2315 2320 tca gga cag cca cag gcc tca cat ctc cct ggc cag cag atc gcc aca 7008 Ser Gly Gln Pro Gln Ala Ser His Leu Pro Gly Gln Gln Ile Ala Thr 2325 2330 2335 tcc ctt agt aac cag gtg cga tct cca gcc cct gtg cag tct cca cgg 7056 Ser Leu Ser Asn Gln Val Arg Ser Pro Ala Pro Val Gln Ser Pro Arg 2340 2345 2350 ccc caa tcc caa cct cca cat tcc agc ccg tca cca cgg ata caa ccc 7104 Pro Gln Ser Gln Pro Pro His Ser Ser Pro Ser Pro Arg Ile Gln Pro 2355 2360 2365 cag cct tca cca cac cat gtt tca ccc cag act gga acc cct cac cct 7152 Gln Pro Ser Pro His His Val Ser Pro Gln Thr Gly Thr Pro His Pro 2370 2375 2380 gga ctc gca gtc acc atg gcc agc tcc atg gat cag gga cac ctg ggg 7200 Gly Leu Ala Val Thr Met Ala Ser Ser Met Asp Gln Gly His Leu Gly 2385 2390 2395 2400 aac cct gaa cag agt gca atg ctc ccc cag ctg aat acc ccc aac agg 7248 Asn Pro Glu Gln Ser Ala Met Leu Pro Gln Leu Asn Thr Pro Asn Arg 2405 2410 2415 agc gca ctg tcc agt gaa ctg tcc ctg gtt ggt gat acc acg gga gac 7296 Ser Ala Leu Ser Ser Glu Leu Ser Leu Val Gly Asp Thr Thr Gly Asp 2420 2425 2430 aca cta gaa aag ttt gtg gag ggt ttg tag 7326 Thr Leu Glu Lys Phe Val Glu Gly Leu 2435 2440 2 2441 PRT Mus VARIANT (1)...(2441) Xaa = Any Amino Acid 2 Met Ala Glu Asn Leu Leu Asp Gly Pro Pro Asn Pro Lys Arg Ala Lys 1 5 10 15 Leu Ser Ser Pro Gly Phe Ser Ala Asn Asp Asn Thr Asp Phe Gly Ser 20 25 30 Leu Phe Asp Leu Glu Asn Asp Leu Pro Asp Glu Leu Ile Pro Asn Gly 35 40 45 Glu Leu Ser Leu Leu Asn Ser Gly Asn Leu Val Pro Asp Ala Ala Ser 50 55 60 Lys His Lys Gln Leu Ser Glu Leu Leu Arg Gly Gly Ser Gly Ser Ser 65 70 75 80 Ile Asn Pro Gly Ile Gly Asn Val Ser Ala Ser Ser Pro Val Gln Gln 85 90 95 Gly Leu Gly Gly Gln Ala Gln Gly Gln Pro Asn Ser Thr Asn Met Ala 100 105 110 Ser Leu Gly Ala Met Gly Lys Ser Pro Leu Asn Gln Gly Asp Ser Ser 115 120 125 Thr Pro Asn Leu Pro Lys Gln Ala Ala Ser Thr Ser Gly Pro Thr Pro 130 135 140 Pro Ala Ser Gln Ala Leu Asn Pro Gln Ala Gln Lys Gln Val Gly Leu 145 150 155 160 Val Thr Ser Ser Pro Ala Thr Ser Gln Thr Gly Pro Gly Ile Cys Met 165 170 175 Asn Ala Asn Phe Asn Gln Thr His Pro Gly Leu Leu Asn Ser Asn Ser 180 185 190 Gly His Ser Leu Met Asn Gln Ala Gln Gln Gly Gln Ala Gln Val Met 195 200 205 Asn Gly Ser Leu Gly Ala Ala Gly Arg Gly Arg Gly Ala Gly Met Pro 210 215 220 Tyr Pro Ala Pro Ala Met Gln Gly Ala Thr Ser Ser Val Leu Ala Glu 225 230 235 240 Thr Leu Thr Gln Val Ser Pro Gln Met Ala Gly His Ala Gly Leu Asn 245 250 255 Thr Ala Gln Ala Gly Gly Met Thr Lys Met Gly Met Thr Gly Thr Thr 260 265 270 Ser Pro Phe Gly Gln Pro Phe Ser Gln Thr Gly Gly Gln Gln Met Gly 275 280 285 Ala Thr Gly Val Asn Pro Gln Leu Ala Ser Lys Gln Ser Met Val Asn 290 295 300 Ser Leu Pro Ala Phe Pro Thr Asp Ile Lys Asn Thr Ser Val Thr Thr 305 310 315 320 Val Pro Asn Met Ser Gln Leu Gln Thr Ser Val Gly Ile Val Pro Thr 325 330 335 Gln Ala Ile Ala Thr Gly Pro Thr Ala Asp Pro Glu Lys Arg Lys Leu 340 345 350 Ile Gln Gln Gln Leu Val Leu Leu Leu His Ala His Lys Cys Gln Arg 355 360 365 Arg Glu Gln Ala Asn Gly Glu Val Xaa Ala Cys Ser Leu Pro His Cys 370 375 380 Arg Thr Met Lys Asn Val Leu Asn His Met Thr His Cys Gln Ala Pro 385 390 395 400 Lys Ala Cys Gln Val Ala His Cys Ala Ser Ser Arg Gln Ile Ile Ser 405 410 415 His Trp Lys Asn Cys Thr Arg His Asp Cys Pro Val Cys Leu Pro Leu 420 425 430 Lys Asn Ala Ser Asp Lys Arg Asn Gln Gln Thr Ile Leu Gly Ser Pro 435 440 445 Ala Ser Gly Ile Gln Asn Thr Ile Gly Ser Val Gly Ala Gly Gln Gln 450 455 460 Asn Ala Thr Ser Leu Ser Asn Pro Asn Pro Ile Asp Pro Ser Ser Met 465 470 475 480 Gln Arg Ala Tyr Ala Ala Leu Gly Leu Pro Tyr Met Asn Gln Pro Gln 485 490 495 Thr Gln Leu Gln Pro Gln Val Pro Gly Gln Gln Pro Ala Gln Pro Pro 500 505 510 Ala His Gln Gln Met Arg Thr Leu Asn Ala Leu Gly Asn Asn Pro Met 515 520 525 Ser Val Pro Ala Gly Gly Ile Thr Thr Asp Gln Gln Pro Pro Asn Leu 530 535 540 Ile Ser Glu Ser Ala Leu Pro Thr Ser Leu Gly Ala Thr Asn Pro Leu 545 550 555 560 Met Asn Asp Gly Ser Asn Ser Gly Asn Ile Gly Ser Leu Ser Thr Ile 565 570 575 Pro Thr Ala Ala Pro Pro Ser Ser Thr Gly Val Arg Lys Gly Trp His 580 585 590 Glu His Val Thr Gln Asp Leu Arg Ser His Leu Val His Lys Leu Val 595 600 605 Gln Ala Ile Phe Pro Thr Pro Asp Pro Ala Ala Leu Lys Asp Arg Arg 610 615 620 Met Glu Asn Leu Val Ala Tyr Ala Lys Lys Val Glu Gly Asp Met Tyr 625 630 635 640 Glu Ser Ala Asn Ser Arg Asp Glu Tyr Tyr His Leu Leu Ala Glu Lys 645 650 655 Ile Tyr Lys Ile Gln Lys Glu Leu Glu Glu Lys Arg Arg Thr Arg Leu 660 665 670 His Lys Gln Gly Ile Leu Gly Asn Gln Pro Ala Leu Pro Ala Ser Gly 675 680 685 Ala Gln Pro Pro Val Ile Pro Pro Ala Gln Ser Val Arg Pro Pro Asn 690 695 700 Gly Pro Leu Pro Leu Pro Val Asn Arg Met Gln Val Ser Gln Gly Met 705 710 715 720 Asn Ser Phe Asn Pro Met Ser Leu Gly Asn Val Gln Leu Pro Gln Ala 725 730 735 Pro Met Gly Pro Arg Ala Ala Ser Pro Met Asn His Ser Val Gln Met 740 745 750 Asn Ser Met Ala Ser Val Pro Gly Met Ala Ile Ser Pro Ser Arg Met 755 760 765 Pro Gln Pro Pro Asn Met Met Gly Thr His Ala Asn Asn Ile Met Ala 770 775 780 Gln Ala Pro Thr Gln Asn Gln Phe Leu Pro Gln Asn Gln Phe Pro Ser 785 790 795 800 Ser Ser Gly Ala Met Ser Val Asn Ser Val Gly Met Gly Gln Pro Ala 805 810 815 Ala Gln Ala Gly Val Ser Gln Gly Gln Glu Pro Gly Ala Ala Leu Pro 820 825 830 Asn Pro Leu Asn Met Leu Ala Pro Gln Ala Ser Gln Leu Pro Cys Pro 835 840 845 Pro Val Thr Gln Ser Pro Leu His Pro Thr Pro Pro Pro Ala Ser Thr 850 855 860 Ala Ala Gly Met Pro Ser Leu Gln His Pro Thr Ala Pro Gly Met Thr 865 870 875 880 Pro Pro Gln Pro Ala Ala Pro Thr Gln Pro Ser Thr Pro Val Ser Ser 885 890 895 Gly Gln Thr Pro Thr Pro Thr Pro Gly Ser Val Pro Ser Ala Ala Gln 900 905 910 Thr Gln Ser Thr Pro Thr Val Gln Ala Ala Ala Gln Ala Gln Val Thr 915 920 925 Pro Gln Pro Gln Thr Pro Val Gln Pro Pro Ser Val Ala Thr Pro Gln 930 935 940 Ser Ser Gln Gln Gln Pro Thr Pro Val His Thr Gln Pro Pro Gly Thr 945 950 955 960 Pro Leu Ser Gln Ala Ala Ala Ser Ile Asp Asn Arg Val Pro Thr Pro 965 970 975 Ser Thr Val Thr Ser Ala Glu Thr Ser Ser Gln Gln Pro Gly Pro Asp 980 985 990 Val Pro Met Leu Glu Met Lys Thr Glu Val Gln Thr Asp Asp Ala Glu 995 1000 1005 Pro Glu Pro Thr Glu Ser Lys Gly Glu Pro Arg Ser Glu Met Met Glu 1010 1015 1020 Glu Asp Leu Gln Gly Ser Ser Gln Val Lys Glu Glu Thr Asp Thr Thr 1025 1030 1035 1040 Glu Gln Lys Ser Glu Pro Met Glu Val Glu Glu Lys Lys Pro Glu Val 1045 1050 1055 Lys Val Glu Ala Lys Glu Glu Glu Glu Asn Ser Ser Asn Asp Thr Ala 1060 1065 1070 Ser Gln Ser Thr Ser Pro Ser Gln Pro Arg Lys Lys Ile Phe Lys Pro 1075 1080 1085 Glu Glu Leu Arg Gln Ala Leu Met Pro Thr Leu Glu Ala Leu Tyr Arg 1090 1095 1100 Gln Asp Pro Glu Ser Leu Pro Phe Arg Gln Pro Val Asp Pro Gln Leu 1105 1110 1115 1120 Leu Gly Ile Pro Asp Tyr Phe Asp Ile Val Lys Asn Pro Met Asp Leu 1125 1130 1135 Ser Thr Ile Lys Arg Lys Leu Asp Thr Gly Gln Tyr Gln Glu Pro Trp 1140 1145 1150 Gln Tyr Val Asp Asp Val Arg Leu Met Phe Asn Asn Ala Trp Leu Tyr 1155 1160 1165 Asn Arg Lys Thr Ser Arg Val Tyr Lys Phe Cys Ser Lys Leu Ala Glu 1170 1175 1180 Val Phe Glu Gln Glu Ile Asp Pro Val Met Gln Ser Leu Gly Tyr Cys 1185 1190 1195 1200 Cys Gly Arg Lys Tyr Glu Phe Ser Pro Gln Thr Leu Cys Cys Tyr Gly 1205 1210 1215 Lys Gln Leu Cys Thr Ile Pro Arg Asp Ala Ala Tyr Tyr Ser Tyr Gln 1220 1225 1230 Asn Arg Tyr His Phe Cys Gly Lys Cys Phe Thr Glu Ile Gln Gly Glu 1235 1240 1245 Asn Val Thr Leu Gly Asp Asp Pro Ser Gln Pro Gln Thr Thr Ile Ser 1250 1255 1260 Lys Asp Gln Phe Glu Lys Lys Lys Asn Asp Thr Leu Asp Pro Glu Pro 1265 1270 1275 1280 Phe Val Asp Cys Lys Glu Cys Gly Arg Lys Met His Gln Ile Cys Val 1285 1290 1295 Leu His Tyr Asp Ile Ile Trp Pro Ser Gly Phe Val Cys Asp Asn Cys 1300 1305 1310 Leu Lys Lys Thr Gly Arg Pro Arg Lys Glu Asn Lys Phe Ser Ala Lys 1315 1320 1325 Arg Leu Gln Thr Thr Arg Leu Gly Asn His Leu Glu Asp Arg Val Asn 1330 1335 1340 Lys Phe Leu Arg Arg Gln Asn His Pro Glu Ala Gly Glu Val Phe Val 1345 1350 1355 1360 Arg Val Val Ala Ser Ser Asp Lys Thr Val Glu Val Lys Pro Gly Met 1365 1370 1375 Lys Ser Arg Phe Val Asp Ser Gly Glu Met Ser Glu Ser Phe Pro Tyr 1380 1385 1390 Arg Thr Lys Ala Leu Phe Ala Phe Glu Glu Ile Asp Gly Val Asp Val 1395 1400 1405 Cys Phe Phe Gly Met His Val Gln Asp Thr Ala Leu Ile Ala Pro His 1410 1415 1420 Gln Ile Gln Gly Cys Val Tyr Ile Ser Tyr Leu Asp Ser Ile His Phe 1425 1430 1435 1440 Phe Arg Pro Arg Cys Leu Arg Thr Ala Val Tyr His Glu Ile Leu Ile 1445 1450 1455 Gly Tyr Leu Glu Tyr Val Lys Lys Leu Val Tyr Val Thr Ala His Ile 1460 1465 1470 Trp Ala Cys Pro Pro Ser Glu Gly Asp Asp Tyr Ile Phe His Cys His 1475 1480 1485 Pro Pro Asp Gln Lys Ile Pro Lys Pro Lys Arg Leu Gln Glu Trp Tyr 1490 1495 1500 Lys Lys Met Leu Asp Lys Ala Phe Ala Glu Arg Ile Ile Asn Asp Tyr 1505 1510 1515 1520 Lys Asp Ile Phe Lys Gln Ala Asn Glu Asp Arg Leu Thr Ser Ala Lys 1525 1530 1535 Glu Leu Pro Tyr Phe Glu Gly Asp Phe Trp Pro Asn Val Leu Glu Glu 1540 1545 1550 Ser Ile Lys Glu Leu Glu Gln Glu Glu Glu Glu Arg Lys Lys Glu Glu 1555 1560 1565 Ser Thr Ala Ala Ser Glu Thr Pro Glu Gly Ser Gln Gly Asp Ser Lys 1570 1575 1580 Asn Ala Lys Lys Lys Asn Asn Lys Lys Thr Asn Lys Asn Lys Ser Ser 1585 1590 1595 1600 Ile Ser Arg Ala Asn Lys Lys Lys Pro Ser Met Pro Asn Val Ser Asn 1605 1610 1615 Asp Leu Ser Gln Lys Leu Tyr Ala Thr Met Glu Lys His Lys Glu Val 1620 1625 1630 Phe Phe Val Ile His Leu His Ala Gly Pro Val Ile Ser Thr Gln Pro 1635 1640 1645 Pro Ile Val Asp Pro Asp Pro Leu Leu Ser Cys Asp Leu Met Asp Gly 1650 1655 1660 Arg Asp Ala Phe Leu Thr Leu Ala Arg Asp Lys His Trp Glu Phe Ser 1665 1670 1675 1680 Ser Leu Arg Arg Ser Lys Trp Ser Thr Leu Cys Met Leu Val Glu Leu 1685 1690 1695 His Thr Gln Gly Gln Asp Arg Phe Val Tyr Thr Cys Asn Glu Cys Lys 1700 1705 1710 His His Val Glu Thr Arg Trp His Cys Thr Val Cys Glu Asp Tyr Asp 1715 1720 1725 Leu Cys Ile Asn Cys Tyr Asn Thr Lys Ser His Thr His Lys Met Val 1730 1735 1740 Lys Trp Gly Leu Gly Leu Asp Asp Glu Gly Ser Ser Gln Gly Glu Pro 1745 1750 1755 1760 Gln Ser Lys Ser Pro Gln Glu Ser Arg Arg Leu Ser Ile Gln Arg Cys 1765 1770 1775 Ile Gln Ser Leu Val His Ala Cys Gln Cys Arg Asn Ala Asn Cys Ser 1780 1785 1790 Leu Pro Ser Cys Gln Lys Met Lys Arg Val Val Gln His Thr Lys Gly 1795 1800 1805 Cys Lys Arg Lys Thr Asn Gly Gly Cys Pro Val Cys Lys Gln Leu Ile 1810 1815 1820 Ala Leu Cys Cys Tyr His Ala Lys His Cys Gln Glu Asn Lys Cys Pro 1825 1830 1835 1840 Val Pro Phe Cys Leu Asn Ile Lys His Asn Val Arg Gln Gln Gln Ile 1845 1850 1855 Gln His Cys Leu Gln Gln Ala Gln Leu Met Arg Arg Arg Met Ala Thr 1860 1865 1870 Met Asn Thr Arg Asn Val Pro Gln Gln Ser Leu Pro Ser Pro Thr Ser 1875 1880 1885 Ala Pro Pro Gly Thr Pro Thr Gln Gln Pro Ser Thr Pro Gln Thr Pro 1890 1895 1900 Gln Pro Pro Ala Gln Pro Gln Pro Ser Pro Val Asn Met Ser Pro Ala 1905 1910 1915 1920 Gly Phe Pro Asn Val Ala Arg Thr Gln Pro Pro Thr Ile Val Ser Ala 1925 1930 1935 Gly Lys Pro Thr Asn Gln Val Pro Ala Pro Pro Pro Pro Ala Gln Pro 1940 1945 1950 Pro Pro Ala Ala Val Glu Ala Ala Arg Gln Ile Glu Arg Glu Ala Gln 1955 1960 1965 Gln Gln Gln His Leu Tyr Arg Ala Asn Ile Asn Asn Gly Met Pro Pro 1970 1975 1980 Gly Arg Asp Gly Met Gly Thr Pro Gly Ser Gln Met Thr Pro Val Gly 1985 1990 1995 2000 Leu Asn Val Pro Arg Pro Asn Gln Val Ser Gly Pro Val Met Ser Ser 2005 2010 2015 Met Pro Pro Gly Gln Trp Gln Gln Ala Pro Ile Pro Gln Gln Gln Pro 2020 2025 2030 Met Pro Gly Met Pro Arg Pro Val Met Ser Met Gln Ala Gln Ala Ala 2035 2040 2045 Val Ala Gly Pro Arg Met Pro Asn Val Gln Pro Asn Arg Ser Ile Ser 2050 2055 2060 Pro Ser Ala Leu Gln Asp Leu Leu Arg Thr Leu Lys Ser Pro Ser Ser 2065 2070 2075 2080 Pro Gln Gln Gln Gln Gln Val Leu Asn Ile Leu Lys Ser Asn Pro Gln 2085 2090 2095 Leu Met Ala Ala Phe Ile Lys Gln Arg Thr Ala Lys Tyr Val Ala Asn 2100 2105 2110 Gln Pro Gly Met Gln Pro Gln Pro Gly Leu Gln Ser Gln Pro Gly Met 2115 2120 2125 Gln Pro Gln Pro Gly Met His Gln Gln Pro Ser Leu Gln Asn Leu Asn 2130 2135 2140 Ala Met Gln Ala Gly Val Pro Arg Pro Gly Val Pro Pro Pro Gln Pro 2145 2150 2155 2160 Ala Met Gly Gly Leu Asn Pro Gln Gly Gln Ala Leu Asn Ile Met Asn 2165 2170 2175 Pro Gly His Asn Pro Asn Met Thr Asn Met Asn Pro Gln Tyr Arg Glu 2180 2185 2190 Met Val Arg Arg Gln Leu Leu Gln His Gln Gln Gln Gln Gln Gln Gln 2195 2200 2205 Gln Gln Gln Gln Gln Gln Gln Gln Asn Ser Ala Ser Leu Ala Gly Gly 2210 2215 2220 Met Ala Gly His Ser Gln Phe Gln Gln Pro Gln Gly Pro Gly Gly Tyr 2225 2230 2235 2240 Ala Pro Ala Met Gln Gln Gln Arg Met Gln Gln His Leu Pro Ile Gln 2245 2250 2255 Gly Ser Ser Met Gly Gln Met Ala Ala Pro Met Gly Gln Leu Gly Gln 2260 2265 2270 Met Gly Gln Pro Gly Leu Gly Ala Asp Ser Thr Pro Asn Ile Gln Gln 2275 2280 2285 Ala Leu Gln Gln Arg Ile Leu Gln Gln Gln Gln Met Lys Gln Gln Ile 2290 2295 2300 Gly Ser Pro Gly Gln Pro Asn Pro Met Ser Pro Gln Gln His Met Leu 2305 2310 2315 2320 Ser Gly Gln Pro Gln Ala Ser His Leu Pro Gly Gln Gln Ile Ala Thr 2325 2330 2335 Ser Leu Ser Asn Gln Val Arg Ser Pro Ala Pro Val Gln Ser Pro Arg 2340 2345 2350 Pro Gln Ser Gln Pro Pro His Ser Ser Pro Ser Pro Arg Ile Gln Pro 2355 2360 2365 Gln Pro Ser Pro His His Val Ser Pro Gln Thr Gly Thr Pro His Pro 2370 2375 2380 Gly Leu Ala Val Thr Met Ala Ser Ser Met Asp Gln Gly His Leu Gly 2385 2390 2395 2400 Asn Pro Glu Gln Ser Ala Met Leu Pro Gln Leu Asn Thr Pro Asn Arg 2405 2410 2415 Ser Ala Leu Ser Ser Glu Leu Ser Leu Val Gly Asp Thr Thr Gly Asp 2420 2425 2430 Thr Leu Glu Lys Phe Val Glu Gly Leu 2435 2440 3 15 PRT Mus 3 Lys Val Glu Gly Asp Met Tyr Glu Ser Ala Asn Ser Arg Asp Glu 1 5 10 15 4 17 DNA Saccharomyces cerevisiae 4 cggaggactg tcctccg 17 

That which is claimed is:
 1. An isolated nucleic acid comprising a sequence encoding a fragment of CREB binding protein (CBP), wherein said fragment includes all or a portion of CBP which binds to CREB.
 2. The isolated nucleic acid of claim 1 wherein said fragment includes the arginine residue at position 600 of SEQ ID NO:2
 3. The isolated nucleic acid of claim 1 wherein said fragment of CBP is 1-661 of SEQ ID NO:2.
 4. The isolated nucleic acid of claim 1 wherein said fragment of CBP is 461-661 of SEQ ID NO:2.
 5. The isolated nucleic acid of claim 1 wherein said fragment of CBP is 634-648 of SEQ ID NO:2
 6. An isolated nucleic acid comprising a sequence encoding a mutant fragment of CREB binding protein (CBP), wherein said fragment includes all or a portion of CBP which binds to CREB.
 7. The isolated nucleic acid of claim 1 wherein said fragment the residue at position 600 of SEQ ID NO:2 is an amino acid other than arginine.
 8. The isolated nucleic acid of claim 1 wherein said fragment includes the residue at position 600 of SEQ ID NO:2 is glutamine.
 9. A CBP fragment encoded by the sequence of claim
 1. 10. An antibody reactive with the CBP fragment of claim
 9. 